Kisspeptin, identified as a natural ligand of GPR54 in 2001, is now considered as a master regulator of puberty and subsequent reproductive functions in mammals. Our previous studies using Kiss1 knockout (KO) rats clearly demonstrated the indispensable role of kisspeptin in gonadotropin-releasing hormone (GnRH)/gonadotropin secretion. In addition, behavioral analyses of Kiss1 KO rats revealed an organizational effect of kisspeptin on neural circuits controlling sexual behaviors. Our studies using transgenic mice carrying a region-specific Kiss1 enhancer-driven reporter gene provided a clue as to the mechanism by which estrogen regulates Kiss1 expression in hypothalamic kisspeptin neurons. Analyses of Kiss1 expression and gonadotropin secretion during the pubertal transition shed light on the mechanism triggering GnRH/gonadotropin secretion at the onset of puberty in rats. Here, we summarize data obtained from the aforementioned studies and revisit the physiological roles of kisspeptin in the mechanism underlying reproductive functions in mammals.

Cover Story:The review article by Uenoyama et al. describes the roles of hypothalamic kisspeptin in the central mechanism regulating puberty and subsequent reproductive functions in mammals. The schematic illustration shows a possible mechanism regulating the pubertal augmentation of Kiss1 expression in the arcuate nucleus (ARC) to trigger pulsatile GnRH/gonadotropin secretion in rodents. The authors suspect that estrogen strongly suppresses ARC Kiss1 expression during the prepubertal period via direct and indirect pathways and that the sensitivity to estrogen negative feedback action on ARC Kiss1 expression decreases during the pubertal transition. The resultant increase in secretion of kisspeptin would trigger GnRH/gonadotropin secretion at pubertal onset (Uenoyama et al. The role of kisspeptin in the mechanism underlying reproductive function in mammals. pp. 469–476).

Sperm freeze-drying is a revolutionary technique, which has been gaining prominence in recent years. The first related significant result was Wakayama and Yanagimachi’s demonstration in 1998 of the birth of healthy mouse offspring by Intracytoplasmic Sperm Injection (ICSI), using epididymal freeze-dried spermatozoa. Mouse, rat, and hamster models were the first small mammals born from lyophilized epididymal spermatozoa, whereas most other studies in this field used ejaculated spermatozoa. In this work, we applied this technique to ram epididymal spermatozoa, checking the correlation between DNA integrity and embryo development following ICSI. To do this, epididymal sperm from four rams was lyophilized in a trehalose, glucose, KCl, HEPES, and Trolox media. To evaluate DNA damage and fragmentation after rehydration, samples were processed for Sperm Chromatin Dispersion test (SCD), Two-Tailed Comet Assay, and were used for ICSI. Ram #2 had a higher rate of spermatozoa with intact DNA compared with rams #1, #3, and #4 (28% vs. 3.8%, 2.8%, and 5%, respectively) and the lowest rate of Single-Strand Breaks (SSBs) (70% vs. 95.9%, 92.6%, and 93% respectively). Ram #3 had a higher level of Double-Strand Breaks (DSBs) compared to Ram #1 (4.6% vs. 0.33%, respectively). Embryo development to the blastocyst stage following ICSI was only reached from rams whose sperm had higher level of intact DNA – Rams #2 and #4 (6%, 5/147 and 6.3%, 4/64, respectively). Definitively, the impact of sperm DNA damage on embryonic development depends on the balance between sperm DNA fragmentation extent, fragmentation type (SSBs or DSBs), and the oocyte’s repair capacity.

Cover Story:Dry biobanking has many advantages over the current paradigm of storing cryopreserved cells under liquid nitrogen. During drying, however, the cells become damaged. The highly condensed spermatozoa DNA has been shown in many desiccation studies to generally maintain its integrity. Using ram freeze-dried epididymal spermatozoa as a model, Palazzese et al. were the first to evaluate both single- and double-strand DNA breaks (SSBs and DSBs, respectively), showing that drying causes minimal DSBs but extensive SSBs (Palazzese L et al., 2018. DNA fragmentation in epididymal freeze-dried ram spermatozoa impairs embryo development, pp. 393–400). Furthermore, the authors also demonstrated that spermatozoa capable of directing embryo development to the blastocyst stage in vitro originated from rams with the least DNA damage Overall, the impact of sperm DNA damage on embryonic development depends on a balance between the extent of sperm DNA fragmentation, fragmentation type, and the oocyte’s repair capacity.

Oog1, an oocyte-specific gene that encodes a protein of 425 amino acids, is present in five copies on mouse chromosomes 4 and 12. In mouse oocytes, Oog1 mRNA expression begins at embryonic day 15.5 and almost disappears by the late two-cell stage. Meanwhile, OOG1 protein is detectable in oocytes in ovarian cysts and disappears by the four-cell stage; the protein is transported to the nucleus in late one-cell to early two-cell stage embryos. In this study, we examined the role of Oog1 during oogenesis in mice. Oog1 RNAi-transgenic mice were generated by expressing double-stranded hairpin Oog1 RNA, which is processed into siRNAs targeting Oog1 mRNA. Quantitative RT-PCR revealed that the amount of Oog1 mRNA was dramatically reduced in oocytes obtained from Oog1-knockdown mice, whereas the abundance of spermatogenesis-associated transcripts (Klhl10, Tekt2, Tdrd6, and Tnp2) was increased in Oog1 knockdown ovaries. Tdrd6 is involved in the formation of the chromatoid body, Tnp2 contributes to the formation of sperm heads, Tekt2 is required for the formation of ciliary and flagellar microtubules, and Klhl10 plays a key role in the elongated sperm differentiation. These results indicate that Oog1 down-regulates the expression of spermatogenesis-associated genes in female germ cells, allowing them to develop normally into oocytes.

Cover Story: Oog1, an oocyte-specific gene, encodes the protein belonging to the leucine-rich repeat (LRR) superfamily. LRR is a motif involved in protein-protein interactions. Complete knockout of Oog1 is challenging because five copies of the Oog1 gene are present on chromosomes 4 and 12. Honda et al. generated Oog1 RNA interference (RNAi)-transgenic mice to investigate the effects of Oog1 knockdown on gene expression in the oocytes (Honda et al. Oocyte-specific gene Oog1 suppresses the expression of spermatogenesis-specific genes in oocytes, pp. 297–301). The abundance of spermatogenesis-specific transcripts was elevated in the Oog1 knockdown ovaries. In addition, a few abnormal oocytes were observed in 6-month-old Oog1 knockdown mouse ovaries. These findings suggested that OOG1 suppresses the expression of spermatogenesis-specific genes in the oocytes and plays important roles during oogenesis.